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Abstract

Recent studies have demonstrated the existence of osteoblast progenitor cells in circulating blood. Here we show that local delivery of bone morphogenetic protein-2 (BMP-2) to cell transplantation sites induces in situ osteogenic differentiation of transplanted human peripheral blood mononuclear cells (PBMNCs) and enhances in vivo bone formation mediated by PBMNC transplantation. Human PBMNCs were seeded on scaffolds with or without BMP-2 and implanted subcutaneously into athymic mice. Nonseeded scaffolds with BMP-2 were also implanted. Eight weeks later, radiographic and histological analyses showed that the PBMNC + BMP-2 group had undergone much more extensive bone formation than either the PBMNC group or BMP-2 group. Only the PBMNC + BMP-2 group expressed human Cbfa1, osteonectin, and osteocalcin, suggesting in situ osteogenic differentiation of and bone formation by transplanted human PBMNCs, while the other groups did not express these genes. This study provides a method to enhance human PBMNC transplantation-mediated bone formation.

Keywords

Bone formation Peripheral blood mononuclear cells (PBMNCs)Bone morphogenetic protein-2 (BMP-2)

Introduction

Peripheral blood mononuclear cells (PBMNCs) contain peripheral blood mesenchymal stem/progenitor cells (PBMSPCs) that have the potential to differentiate into many cell types including chondrocytes, adipocytes, myoblasts, and osteoclasts (18,24). Recently, several studies have demonstrated the existence of osteoblast lineage cells or osteoblast progenitor cells (OPCs) in circulating blood (6,13,27,28,32). OPCs are mobilized from bone marrow into peripheral blood (27), migrate to injured bone tissue by chemoattraction of stromal cell-derived factor-1, and differentiate into osteoblasts by bone morphogenetic protein-2 (BMP-2) (27,28). Osteoblast lineage cells in the circulating blood were shown to form mineralized nodules in vitro and bone in vivo (6).

OPCs from peripheral blood can be used to regenerate bone. In a previous study, bone tissue was formed in vivo by seeding CD45-negative PBMNCs on scaffolds, differentiating the cells in vitro with an osteogenic medium for 2 weeks, and then transplanting the differentiated cells subcutaneously into nude mice (28). Transplantation of undifferentiated PBMNCs did not lead to bone formation in vivo (28). However, this method cannot be used to treat patients in emergency situations due to the requirement of in vitro culture for osteogenic differentiation of OPCs for 2 weeks, and would increase treatment costs. Furthermore, isolation and culture of PBMSPCs or OPCs seems difficult, as some attempts have failed to isolate and culture PBMSPCs from circulating blood (20,34).

If undifferentiated PBMNCs transplanted for osteogenic differentiation without in vitro culture could regenerate bone in vivo, this method could be used to treat patients in emergency applications and reduce treatment costs. Thus, we hypothesized that local delivery of BMP-2 to the cell transplantation site induces in situ osteogenic differentiation of human PBMNCs transplanted in an undifferentiated state and enhances in vivo bone formation. To test this hypothesis, human PBMNCs were seeded on scaffolds with or without BMP-2 and implanted subcutaneously into athymic mice. Nonseeded scaffolds with BMP-2 were also implanted. Eight weeks later, radiographic and histological analyses were performed to evaluate in vivo bone formation. Expression of human core-binding factor α1 (Cbfa1), osteonectin, and osteocalcin was evaluated to determine whether transplanted human PBMNCs underwent in situ osteogenic differentiation and contributed to bone formation.

Materials and Methods

Human PBMNC Isolation

Whole peripheral blood (400 ml of whole blood/each person) was obtained from two healthy volunteers (22 years old) by venipuncture. The whole blood was centrifuged on a Ficoll-Paque density gradient (specific gravity = 1.077, Amersham Biosciences, Arlington Heights, IL, USA) for 30 min at 230 × g. PBMNCs were isolated from the buffy coat layer between the Ficoll-Paque regent and blood plasma component and washed three times in 10 ml of warm Dulbecco's phosphate-buffered saline (Sigma, St. Louis, MO, USA) (2). Subsequently, PBMNCs were counted with a hematocytometer and used immediately. The use of human blood for this study was approved by the Institutional Review Board at Seoul National University (^#CRI10020).

Preparation of PGA/PLGA Mesh and Fibrin Gel

Unwoven polyglycolic acid (PGA) fibers (5 × 5 mm, 2 mm in thickness, Albani International Inc., Albany, NY, USA) were stabilized by immersing them in a poly-(lactic-co-glycolic acid) (PLGA 75/25, Lakeshore Biomaterials, AL, USA) solution (5% in methyl chloride) for 2 s and then allowed to incubate in air for 1 h (14). The PGA/PLGA meshes were washed three times with PBS and lyophilized for 48 h for removed residual organic solvent. The PGA/PLGA meshes were immersed in the Dulbecco's modified Eagle's medium (DMEM, Gibco, NY, USA) containing 10% (v/v) fetal bovine serum (Gibco) and 1% penicillin/streptomycin for 30 min to allow the adsorption of cell adhesion proteins (e.g., fibronectin) contained in the serum onto the mesh (15). The excessive medium left within the meshes was removed by extensive suction before cell seeding.

Fibrin matrix was prepared from a commercially available fibrin gel kit (Greenplast^®, Greencross Co., Yongin, Korea) (1). Plasminogen-free fibrinogen (100 mg) and fibrin-stabilizing factor XIII (66 units) were dissolved in 1 ml of plasmin inhibitor aprotinin solution (1100 kIU/ml) for fibrinogen solution. Thrombin (400–600 IU) was dissolved in 1 ml of calcium chloride solution (5.9 mg/ml) for the thrombin solution.

In Vivo Transplantation

Athymic mice (BALB/c-nu, 7 weeks old, female, SLC, Tokyo, Japan) were anesthetized with an intramuscular administration of ketamine hydrochloride (50 mg/kg, Yuhan Co., Seoul, Korea) and xylazine hydrochloride (5 mg/kg, Bayer Korea Ltd., Seoul, Korea). Fibrinogen solution containing BMP-2 (1 μg per scaffold, R&D Systems, Minneapolis, MN, USA) or no BMP-2 and thrombin solution containing PBMNCs (3 × 10⁶ cells per scaffold) were mixed at a 1:1 volume ratio, and added on PGA/PLGA mesh scaffolds (50 μl each). The BMP-2 dose of 1 μg was chosen because previous studies showed excellent ectopic bone formation at this dose (11,12). Cell-seeded scaffolds with or without BMP-2 were implanted into the dorsal subcutaneous spaces of the athymic mice. Nonseeded scaffolds containing BMP-2 were also implanted. The animals were sacrificed at 8 weeks after implantation. The time point was chosen because previous studies showed excellent bone formation at this time point (19,35). Ten samples were taken per group. The animal experiments were approved by the Institutional Animal Care and Use Committee at Seoul National University (#SNU-100203-6).

Analysis of Bone Formation

Eight weeks after transplantation, the mice were sacrificed, and the implants (n = 10 per group) were retrieved and fixed in a phosphate-buffered formaldehyde (10% v/v) solution. Bone formation was evaluated with soft X-ray (n = 4 per group), microcomputed tomography (CT) scan (n = 3 per group), and histological analysis. Radiograms of the implants were taken with a soft X-ray apparatus (Softex, Sofron Co., Tokyo, Japan). CT images, each representing 58-μm slice thickness, were obtained from a micro-CT system (SkyScan-1172, Skyscan, Kontich, Belgium).

After radiographic analysis, the each sample was cut in half and used for histological and immunohistochemical analyses (n = 7 per group). The samples were embedded in optimal cutting temperature compound (OCT, TISSUE-TEK 4583, Sakura Finetek USA Inc., Torrance, CA), frozen, and sectioned in 10-μm-thick slices, and examined with von Kossa staining. For histological analysis, the samples were decalcified in 10% buffered nitric acid (Sigma) solution for 4 h. Then samples were embedded in paraffin, sectioned in 4-μm-thick slices, and examined with hematoxylin and eosin (H&E) staining and Goldner's trichrome staining. The area of bone formation was measured using an image analysis system (KS400, Zeiss, Munich, Germany) coupled to a light microscope. The bone formation area was expressed as the percentage of bone area in total cross-sectional area [(bone area/total area) × 100%]. For immunohistochemical analysis, the harvested samples were embedded in OCT compound, frozen, and cut into 10-μm-thick sections at −22°C. Sections of the samples were immunofluorescently stained with anti-osteocalcin (Abcam, Cambridge, UK) and human nucleus histone H1 (HNH H1, Abcam). The staining signal for osteocalcin was visualized with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The staining signal for HNH H1 was visualized with Tetramethyl Rhodamine Iso-Thiocyanate (TRITC)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). The sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) and examined using a fluorescence microscope (Nikon TE2000, Tokyo, Japan).

RT-PCR (n = 3 per group) was performed 8 weeks after implantation. Total RNA was isolated from retrieved implants using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Specimens were lysed and homogenized with 1 ml of TRIzol reagent, added to 200 μl of chloroform, and centrifuged at 12,000 rpm for 15 min. The RNA pellets were washed with 75% (v/v) ethanol, air dried, and dissolved in RNase-free water. Reverse transcription was performed with 5 μg of pure RNA using SuperScript II reverse transcriptase (Invitrogen). Synthesized cDNA was amplified by PCR using the following primers: 1) human Cbfa1—sense 5′-TTCATCCCTCACTGAGAG-3′ and antisense 5′-TCAGCGTCAACACCATCA-3′, 2) human osteonectin—sense 5′-ACTGGCTCAAGAACGTCCTG-3′ and antisense 5′-GAC AGAATCCGGTACTGTGG-3′, 3) human osteocalcin—sense 5′-CGCAGCCACCGAGACACCAT-3′ and antisense 5′-AGGGCAAGGGGAAGAGGAAAGAAG-3′, 4) mouse β-actin—sense 5′-TGGACTTCGAGCAAGAGATGG-3′ and antisense 5′-ATCTCCTTCTGCATCCTGTCG-3′. PCR was carried out for 30 cycles of denaturing (94°C, 30 s), annealing (58°C, 45 s), and extension (72°C, 45 s) with a final extension at 72°C for 10 min. PCR products were visualized by electrophoresis on 1.5% (w/v) agarose gels with ethidium bromide staining and analyzed with a gel documentation system (Gel Doc 1000, Bio-Rad Laboratories, Hercules, CA, USA).

Statistical Analysis

All quantitative data were expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's honestly significant difference (HSD) post hoc test using SPSS software (SPSS Inc., Chicago, IL, USA). Values of p < 0.05 were considered statistically significant.

Results

Radiographic Analysis

At 8 weeks after implantation, soft X-ray radiography and computerized tomography of the implants revealed that the PBMNC + BMP-2 group showed much more extensive in vivo bone formation than either the PBMNC group or BMP-2 group (Fig. 1). The PBMNC group showed nearly no bone formation. The PBMNC + BMP-2 group induced a considerable extent of bone formation, which was much more than that of BMP-2 group.

Figure 1.

Representative soft X-ray and μ-CT (computed tomography) images of implants retrieved 8 weeks after transplantation of peripheral blood mononuclear cells (PBMNCs), bone morphogenetic protein-2 (BMP-2), and PBMNC + BMP-2 groups. Mineralized tissue appeared white. Scale bars: 1 mm.

Histological Analysis

Eight weeks after implantation, histological analysis of the midportion sections of the implants revealed that implantation of PBMNCs alone (PBMNC group) induced a very low extent of bone formation (Fig. 2A). Implantation of BMP-2-loaded scaffolds (BMP-2 group) resulted in considerable bone formation. Importantly, implantation of PBMNCs seeded on BMP-2-loaded scaffolds (PBMNC + BMP-2 group) induced the most bone formation. The area of bone formation in the PBMNC + BMP-2 group was greatest among all groups, and 60-fold greater than the PBMNC group (Fig. 2B).

Figure 2.

(A) Histological analysis of implants retrieved 8 weeks after transplantation of PBMNC, BMP-2, and PBMNC + BMP-2 groups with H&E, Goldner's trichrome, and von Kossa staining. Scale bars: 100 μm. (B) Bone formation area in the implants, as determined with histomorphometry of von Kossa staining (n = 5). a: adipose tissue, b: mature bone, f: fibrous tissue.

Osteogenic Differentiation

Immunohistochemical analysis of implants retrieved 8 weeks after transplantation indicated that transplanted human PBMNCs survived in both the PBMNC and PBMNC + BMP-2 groups at 8 weeks, as HNH N1-positive cells were found in the implants (Fig. 3). Osteocalcin was produced in both the PBMNC group and PBMNC + BMP-2 group, and the production was larger in the PBMNC + BMP-2 group. The merged image in Figure 3 shows that the transplanted PBMNCs underwent osteogenic differentiation in PBMNC + BMP-2 group and did not in PBMNC group.

Figure 3.

Immunohistochemical analysis of implants retrieved 8 weeks after transplantation of PBMNC, BMP-2, and PBMNC + BMP-2 groups. Blue indicates DAPI (nuclei). Red indicates HNH H1 (human nucleus histone H1). Green indicates osteocalcin (OC).

Expressions of human Cbfa1, osteonectin, and osteocalcin in the implants were evaluated to determine whether transplanted human PBMNCs underwent in situ osteogenic differentiation and contributed to the bone formation (Fig. 4). RT-PCR analysis showed that only the PBMNC + BMP-2 group expressed human Cbfa1, osteonectin, and osteocalcin, suggesting in situ osteogenic differentiation of and bone formation by transplanted human PBMNCs. Human Cbfa1, osteonectin, and osteocalcin were not detected in the PBMNC group, suggesting no osteogenic differentiation of transplanted human PBMNCs. Human Cbfa1, osteonectin, and osteocalcin were not detected in the BMP-2 group in which human PBMNCs were not transplanted.

Figure 4.

RT-PCR analysis for osteogenic gene expression in implants retrieved 8 weeks after transplantation of human PBMNC, BMP-2, and human PBMNC + BMP-2 groups. RT-PCR analysis was performed using human-specific primers to detect human gene expression by implanted human PBMNCs.

Discussion

PBMNCs could be a more convenient cell source for bone regeneration than bone marrow-, adipose-, or cord blood-derived stem cells. Current cell-based therapies for bone regeneration use bone marrow-, adipose-, or cord blood-derived stem cells (4,8–10,21,26,36,37). Peripheral blood draws from patients are much less invasive, more comfortable, and safer than isolation of bone marrow and adipose tissue. Compared to allogenic cord blood-derived stem cells, autologous PBMNCs allow patients to avoid immune rejection. Moreover, implantation of PBMNCs that are not osteogenically differentiated would be convenient for patients, because it avoids the in vitro culture process for osteogenic differentiation, which takes several weeks (23) and increases treatment costs of osteogenic differentiation prior to implantation.

The more extensive bone formation in the PBMNC + BMP-2 group than in the PBMNC group (Figs. 1 and 2) is likely due to osteogenic differentiation of human PBMNCs induced by BMP-2 delivered to the cell transplantation sites. In the PBMNC group, the implanted human PBMNCs did not express human Cbfa1, osteonectin, and osteocalcin (Fig. 4), resulting in nearly no bone formation (Figs. 1 and 2). Cbfa1 is a transcriptional activator of osteoblastic differentiation (7). Osteocalcin is an indicator of osteoblastic differentiation, a major component of bone extracellular matrix, and synthesized exclusively by osteoblastic cells in the late stage of maturation (22,31). Osteonectin is the most abundant noncollagen extracellular matrix protein in bone (33). Expression of Cbfa1, oseteonectin, and osteocalcin is observed in mesenchymal cells in bone fracture in mice for at least 21–28 days (3,16). Thus, expression of these factors for at least 21–28 days would be appropriate for effective bone formation. Expression of Cbfa1 and osteonectin and that of osteocalcin (17) initiate 4 days and 5 days after bone fracture in mice, respectively. In the PBMNC + BMP-2 group, BMP-2 delivered to the sites of the PBMNC implantation induced osteoblastic differentiation of the implanted human PBMNCs, as indicated by expression of human Cbfa1, osteonectin, and osteocalcin (Fig. 4), which likely enhanced the bone formation compared to the PBMNC group. The implantation of PBMNCs and BMP-2 may not cause side effects such as tumor risks and uncontrolled self-renewal of undifferentiated cells. BMP-2 is known to stimulate differentiation rather than proliferation. In addition, the BMP-2 dose used in this study (1 μg/50 μl = 20 μg/ml) was far less than the clinically approved dose (ca. 1.5 mg/ml) (30).

BMPs are members of the transforming growth factor-β superfamily that play important roles in most morphogenetic processes during development (25). BMPs stimulate bone formation by inducing the differentiation of mesenchymal progenitor cells into osteoblastic lineage (5). The intracellular events for BMP-2-induced osteogenic differentiation of mesenchymal progenitor cells involve BMP receptor activation, receptor-regulated-Smad (R-Smad) activation, kinase activation, and osteogenic transcription factor expression (29). Binding of BMP-2 to a receptor complexes composed of monomeric BMP type I receptor and BMP type II receptor initiates the Smad pathway by phosphorylation of R-Smad, resulting in the regulation of the osteogenic transcription factors, runt-related transcription factor 2 (Runx2) and Osterix (Osx). The transcription factors stimulate expression of osteoblastic genes, type I collagen, alkaline phosphatase, and osteocalcin. A second pathway for the BMP-2 signaling, which is Smad-independent, involves mitogen-activated protein kinase (MAPK) pathways (25).

The in vivo bone formation observed in the BMP-2 group without PBMNC transplantation (Figs. 1 and 2) was likely caused by migration of host osteogenic stem or progenitor cells from the surrounding tissues to the BMP-2-loaded scaffolds and subsequent osteoblastic differentiation of the host cells induced by the locally delivered BMP-2. BMP-2 released from the scaffolds may diffuse to the surrounding tissue and induce host cell migration and osteoblastic differentiation of the migrating cells. A previous study showed BMP-2 release from fibrin gel for 11 days (35). BMP-2 delivery for up to 21 days would be appropriate for effective bone formation, because BMP-2 expression is observed from day 2 to day 21 in bone fracture in mice (16). The results of a previous study demonstrated that BMP-2 released from scaffolds induced bone marrow-derived OPCs in peripheral blood to regenerate bone in the scaffolds (27). The lower extent of bone formation in the BMP-2 group than in the PBMNC + BMP-2 group (Figs. 1 and 2) could be due to a lower number of OPCs in the scaffolds of the BMP-2 group than the PBMNC + BMP-2 group, although BMP-2 induces recruitment of OPCs from peripheral blood to the scaffolds.

A large portion of bone formation in the PBMNC + BMP-2 group was mediated by transplanted human PBMNCs. The transplanted human PBMNCs survived 8 weeks after transplantation (Fig. 3). The transplanted human PBMNCs in the PBMNC + BMP-2 group differentiated osteogenically as they expressed human Cbfa1, osteonectin, and osteocalcin (Fig. 3). PBMNCs transplanted in an undifferentiated state did not form bone in vivo in the PBMNC group (Figs. 1 and 2). Bone formation was much more extensive in the PBMNC + BMP-2 group than in the BMP-2 group (Figs. 1 and 2). Thus, the difference in bone formation between the PBMNC + BMP-2 and BMP-2 groups was likely due to bone formation by PBMNCs in the PBMNC + BMP-2 group.

Conclusion

Undifferentiated cells could have negative effects such as tumor formation. However, we could not find any immune response and inflammation in transplanted sites. Local delivery of BMP-2 to cell transplantation sites induces in situ osteogenic differentiation of undifferentiated human PBMNCs and enhances in vivo bone formation. The bone generation method developed in this study maybe be a more convenient alternative to current cell-based therapies using bone marrow- or adipose-derived stem cells, because peripheral blood drawing is much less invasive, more comfortable, and safer than isolation of bone marrow and adipose tissue. Moreover, because the method can avoid the in vitro culture process for osteogenic differentiation prior to implantation, patients' waiting period and treatment costs would be reduced.

Footnotes

Acknowledgments

This work was supported by a grant (SC3220) from the Stem Cell Research Center of the 21th Century Frontier Program, the Ministry of Education, Science and Technology, and a grant (2009-0080769, 2010-0020352) from the National Research Foundation of Korea. The authors declare no conflicts of interest.

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Enhancement of Human Peripheral Blood Mononuclear Cell Transplantation-Mediated Bone Formation

Abstract

Keywords

Introduction

Materials and Methods

Human PBMNC Isolation

Preparation of PGA/PLGA Mesh and Fibrin Gel

In Vivo Transplantation

Analysis of Bone Formation

Statistical Analysis

Results

Radiographic Analysis

Histological Analysis

Osteogenic Differentiation

Discussion

Conclusion

Footnotes

Acknowledgments

References